Traditional microscopy permits the examination of biological structures using a variety of techniques based on light transmission, reflection, polarization, luminescence, and scattering. By combining these methods with staining protocols, clinicians and researchers both acquire powerful tools to evaluate biological and other specimens.
With the advent of intense laser light sources, it has become practical to provide specimen images based on both linear and non-linear effects obtained in response to an intense optical beam. For example, with such intense beams, a specimen illuminated by a laser at a first wavelength can produce radiation at a second, third, fourth, or other harmonic of the first wavelength, or at some other wavelengths. In so-called multiphoton microscopy a specimen emits a light flux in response to a light flux exciting a material via a multiphoton process (usually multiphoton absorbance leading to luminescence, e.g., fluorescence or phosphorescence). In fluorescence microscopy, fluorescence produced in a specimen in response to a stimulating light flux is used to form a specimen image. In both fluorescence microscopy and other multiphoton microscopies, there are methods that relay and focus emitted light flux to produce an image directly; usually, however, the image is formed by quantifying the emitted light flux that is localized by scanning the stimulating light flux across the specimen.
Generally, light produced by multiphoton emission (e.g., fluorescence) is relatively weak, and high optical intensities are needed to produce satisfactory emitted light levels. Thus, when nonlinear effects or fluorescence are used in microscopic evaluations, only weak optical signals are often available for imaging and these optical signals must be detected in the presence of much stronger illumination beams. In addition, in order to increase multiphoton emission or fluorescence, increased illumination intensities are needed. Unfortunately, increased illumination beam intensity is associated with specimen damage or degradation such as specimen bleaching. Thus, even though increased illumination intensity can produce increased nonlinear emission and/or fluorescence, the associated damage reduces useful observation times. Accordingly, efficient collection of nonlinear emission beyond that which is available with conventional high numerical aperture objective lenses is needed.
Existing multiphoton microscopy systems may be enhanced by combinations of minors and lenses arranged to collect multiphoton fluorescence, either the component that propagates generally in the direction of the stimulus beam and is transmitted by a specimen (“trans light”) or the multiphoton fluorescence that is propagates backwards toward the excitation beam (“epi” light). One representative system is described in Balaban, Combs, and Knutson, U.S. Pat. No. 7,667,210, that is incorporated herein by reference. While such systems can be practical and permit improved light detection with respect to conventional systems, further improvements are needed.